This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ras is a key regulator of directed cell migration, controlling directional sensing, cell motility as well as signal relay in Dictyostelium. We showed that Ras is preferentially activated at the plasma membrane at the leading edge of migrating cells, resulting in the localized activation of downstream effectors that regulate the actin cytoskeleton and thereby promote directed cell movement. We recently identified a protein complex made of two Ras guanine exchange factors (RasGEFs), which are activators of Ras, a scaffold protein, and the protein phosphatase 2A (PP2A). We found that this RasGEF complex selectively controls an important Ras signaling pathway that regulates both chemotaxis and signal relay. Since activated Ras is locally enriched at the front of migrating cells, we hypothesized that the RasGEFs would be similarly localized. We tested this hypothesis by fusing GFP to different components of the RasGEF complex and assessing their localization in chemotaxing cells under a confocal microscope. We now have evidence that upon sudden uniform chemoattractant stimulation that the RasGEF complex is transiently translocated from the cytoplasm to the plasma membrane. However, because of the strong cytosolic signal, we were unable to determine if the RasGEF complex is present at the plasma membrane in polarized cells that are migrating in a chemoattractant gradient. We were previously faced with a similar problem when studying the localization of a Ras GTPase activating protein (RasGAP), a Ras inhibitor in this case, and TIRF microscopy allowed us to detect plasma membrane localization of this protein when other types of microscopy did not. We are thus confident that TIRF microscopy would also provide us with an answer concerning any plasma membrane localization of the RasGEF complex in chemotaxing cells. All the conditions to look at chemotaxing cells in TIRF microscopy have been optimized already so we expect we will be able to get all of the needed data in only a few sessions. We propose to look at two different cell lines: one expressing a GFP-fused protein from the RasGEF complex and a control cell line expressing soluble, cytosolic GFP.